The present invention relates to a technique of synthesizing and separating a sugar chain. In particular, the present invention relates to a sugar chain synthesizer used to automatically perform such processes.
Complex carbohydrates play important roles in cells, such as information transmission, or intracellular differentiation such as recognition of viruses, cancer cells, or blood types. Clarification of the functions of sugar chains is considered to be a post-genome project. Methods for synthesizing oligonucleic acids or peptides have already been provided and automated. However, a method for synthesizing a sugar chain still has many problems.
In order to clarify the functions of sugar chains, the establishment of a method for synthesizing a sugar chain and realization of an efficient synthesizer have been desired. At present, the following three methods for synthesizing a sugar chain have been applied:                (1) a method of using chemical synthesis;        (2) a fermentation method of using genetically-altered cells or microorganisms; and        (3) a synthetic method of using glycosyltransferase.        
The method in (1) above is complicated and has many reaction steps because sugar chains of interest are successively synthesized while protecting OH groups other than the OH group to be chemically bound. The method in (2) above enables the mass production of sugar chains of interest, but the following purification process becomes complicated. The method in (3) above has been developed to overcome the complexity of the methods in (1) and (2) above, and it is disclosed in JP-A-11-42096 (Patent Document 1) for example. In addition, since the method in (3) above involves a selective synthesis using glycosyltransferase, differing from the method in (1) above, it is not necessary to protect OH groups. Moreover, since this method causes only small quantities of by-products, the purification process following the synthesis is easy.
A sugar chain synthesizer is disclosed in JP-A-5-500905 (Patent Document 2).
When the synthesis of sugar chains is carried out by the method described in (3) above actually using a synthesizer, a batch system is currently applied to successively react a plurality of sugars. In the batch system, separation and purification of a product is carried out in every step, and then, the routine goes to the next reaction. In order to carry out all processes, human power is absolutely necessary.
In the case of the synthesizer disclosed in the aforementioned Patent Document 2, it is necessary to continuously connect reaction columns with separation and purification means in series, depending on the order of sugars to be reacted. That is to say, this synthesizer is problematic in that although the sugars to be reacted are of the same type, reaction columns and separation and purification means are required depending on the number of the sugars to be reacted, and in that the synthesizer should be large-scale equipment. In addition, Patent Document 2 does not describe a method for automatically and continuously synthesizing sugar chains.
It is an object of the present invention to provide a sugar chain synthesizer, in which when a plurality of sugars are successively reacted, a single reactor and one or more separation means are used, and reaction products or glycosyltransferases are recovered and recycled, so as to continuously carry out every reaction steps.
In order to achieve the aforementioned object, the sugar chain synthesizer of the present invention is characterized in that it comprises: a pump for supplying a buffer solution; a plurality of vessels containing respective sugar nucleotide solutions; a plurality of vessels containing respective glycosyltransferases; a reactor containing a primer that is a water-soluble polymer, into which the above described sugar nucleotide solutions and glycosyltransferases are introduced; a sampling means for sampling the solution contained in the above described reactor and introducing it into a flow channel through which the above described buffer solution flows; a first ultrafilter for subjecting the solution introduced by the above described sampling means to ultrafiltration; a second ultrafilter for subjecting an effluent from the above described first ultrafilter to ultrafiltration; a first selector valve, which is provided between the above described first and second ultrafilters and comprises a flow channel for returning the effluent from the above described first ultrafilter to the above described reactor and a flow channel for supplying the effluent to the above described second ultrafilter; and a second selector valve, which is provided downstream of the above described second ultrafilter and comprises a flow channel for delivering the effluent from the above described second ultrafilter to a drain and a flow channel for supplying the effluent to the above described vessel containing respective glycosyltransferase.
In addition, the sugar chain synthesizer of the present invention is further characterized in that it comprises: a pump for supplying a buffer solution; a plurality of vessels containing respective sugar nucleotide solutions; a plurality of vessels containing respective glycosyltransferases; a reactor containing a primer that is a water-soluble polymer, into which the above described sugar nucleotide solutions and glycosyltransferases are introduced; a sampling means for sampling the solution contained in the above described reactor and introducing it into a flow channel through which the above described buffer solution flows; a GPC column for separating the solution introduced by the above described sampling means depending on the molecular weight; first and second ultrafilters, into which an eluant from the above described GPC column is introduced; a first selector valve, which is provided between the above described GPC column and the above described first and second ultrafilters, and selectively supplies the eluant from the above described GPC column to the above described first ultrafilter or the above described second ultrafilter; a second selector valve, which is provided downstream of the above described first ultrafilter and comprises a flow channel for supplying an effluent from the above described first ultrafilter to the above described reactor and a flow channel for delivering the effluent to a drain; and a third selector valve, which is provided downstream of the above described second ultrafilter and comprises a flow channel for delivering the effluent from the above described first ultrafilter to the above described drain and a flow channel for supplying the effluent to the above described vessel containing respective glycosyltransferase.
According to the present invention, it becomes possible to return an effluent (reaction product) that had once passed through a reactor and a separation column to the reactor and the separation column again. By repeating such a process, it becomes possible to continuously and automatically carry out a complicated synthesis of sugar chains.
Other objects, features and advantages of the invention will become apparent from the following description of the embodiments of the invention taken in conjunction with the accompanying drawings.